Comparison of three 18F-labeled 2-nitroimidazoles for imaging hypoxia in breast cancer xenografts: [18F]FBNA, [18F]FAZA and [18F]FMISO.

BACKGROUND: Tumour hypoxia is associated with increased metastasis, invasion, poor therapy response and prognosis. Most PET radiotracers developed and used for clinical hypoxia imaging belong to the 2-nitroimidazole family. Recently we have developed novel 2-nitroimidazole-derived PET radiotracer [18F]FBNA (N-(4-[18F]fluoro-benzyl)-2-(2-nitro-1H-imidazol-1-yl)-acet-amide), an 18F-labeled analogue of antiparasitic drug benznidazole. The present study aimed to analyze its radio-pharmacological properties and systematically compare its PET imaging profiles with [18F]FMISO and [18F]FAZA in preclinical triple-negative (MDA-MB231) and estrogen receptor-positive (MCF-7) breast cancer models. METHODS: In vitro cellular uptake experiments were carried out in MDA-MB321 and MCF-7 cells under normoxic and hypoxic conditions. Metabolic stability in vivo was determined in BALB/c mice using radio-TLC analysis. Dynamic PET experiments over 3 h post-injection were performed in MDA-MB231 and MCF-7 tumour-bearing mice. Those PET data were used for kinetic modelling analysis utilizing the reversible two-tissue-compartment model. Autoradiography was carried out in tumour tissue slices and compared to HIF-1α immunohistochemistry. Detailed ex vivo biodistribution was accomplished in BALB/c mice, and this biodistribution data were used for dosimetry calculation. RESULTS: Under hypoxic conditions in vitro cellular uptake was elevated in both cell lines, MCF-7 and MDA-MB231, for all three radiotracers. After intravenous injection, [18F]FBNA formed two radiometabolites, resulting in a final fraction of 65 ± 9 % intact [18F]FBNA after 60 min p.i. After 3 h p.i., [18F]FBNA tumour uptake reached SUV values of 0.78 ± 0.01 in MCF-7 and 0.61 ± 0.04 in MDA-MB231 tumours (both n = 3), representing tumour-to-muscle ratios of 2.19 ± 0.04 and 1.98 ± 0.15, respectively. [18F]FMISO resulted in higher tumour uptakes (SUV 1.36 ± 0.04 in MCF-7 and 1.23 ± 0.08 in MDA-MB231 (both n = 4; p < 0.05) than [18F]FAZA (0.66 ± 0.11 in MCF-7 and 0.63 ± 0.14 in MDA-MB231 (both n = 4; n.s.)), representing tumour-to-muscle ratios of 3.24 ± 0.30 and 3.32 ± 0.50 for [18F]FMISO, and 2.92 ± 0.74 and 3.00 ± 0.42 for [18F]FAZA, respectively. While the fraction per time of radiotracer entering the second compartment (k3) was similar within uncertainties for all three radiotracers in MDA-MB231 tumours, it was different in MCF-7 tumours. The ratios k3/(k3 + k2) and K1*k3/(k3 + k2) in MCF-7 tumours were also significantly different, indicating dissimilar fractions of radiotracer bound and trapped intracellularly: K1*k3/(k2 + k3) [18F]FMISO (0.0088 ± 0.001)/min, n = 4; p < 0.001) > [18F]FAZA (0.0052 ± 0.002)/min, n = 4; p < 0.01) > [18F]FBNA (0.003 ± 0.001)/min, n = 3). In contrast, in MDA-MB231 tumours, only K1 was significantly elevated for [18F]FMISO. However, this did not result in significant differences for K1*k3/(k2 + k3) for all three 2-nitroimidazoles in MDA-MB231 tumours. CONCLUSION: Novel 2-nitroimidazole PET radiotracer [18F]FBNA showed uptake into hypoxic breast cancer cells and tumour tissue presumably associated with elevated HIF1-α expression. Systematic comparison of PET imaging performance with [18F]FMISO and [18F]FAZA in different types of preclinical breast cancer models revealed a similar tumour uptake profile for [18F]FBNA with [18F]FAZA and, despite its higher lipophilicity, still a slightly higher muscle tissue clearance compared to [18F]FMISO.

Chlordiazepoxide-induced delirium in a patient undergoing alcohol withdrawal: a case report.

BACKGROUND: Ethanol dependence is associated with a discontinuation withdrawal delirium. Chlordiazepoxide is frequently successfully used in its treatment. CASE PRESENTATION: A 27-year-old, Caucasian female with ethanol dependence who had objective symptoms of withdrawal experienced worsening of her delirium after administration of chlordiazepoxide, but improved with lorazepam and cleared with discontinuation of benzodiazepine administration. CONCLUSIONS: Worsening of delirium appears to be related to the specific use of chlordiazepoxide, but the mechanism of this effect is not clear. While this case does not alter the standard care of ethanol dependence, it does alert clinicians that our treatment approach may not be fully benign.

Synthesis of a 2-nitroimidazole derivative N-(4-[18F]fluorobenzyl)-2-(2-nitro-1H-imidazol-1-yl)-acetamide ([18 F]FBNA) as PET radiotracer for imaging tumor hypoxia.

BACKGROUND: Tissue hypoxia is a pathological condition characterized by reducing oxygen supply. Hypoxia is a hallmark of tumor environment and is commonly observed in many solid tumors. Non-invasive imaging techniques like positron emission tomography (PET) are at the forefront of detecting and monitoring tissue hypoxia changes in vivo. RESULTS: We have developed a novel 18F-labeled radiotracer for hypoxia PET imaging based on cytotoxic agent benznidazole. Radiotracer N-(4-[18F]fluorobenzyl)-2-(2-nitro-1H-imidazol-1-yl)acetamide ([18F]FBNA) was synthesized through acylation chemistry with readily available 4-[18F]fluorobenzyl amine. Radiotracer [18F]FBNA was obtained in good radiochemical yields (47.4 ± 5.3%) and high radiochemical purity (> 95%). The total synthesis time was 100 min, including HPLC purification and the molar activity was greater than 40 GBq/µmol. Radiotracer [18F]FBNA was stable in saline and mouse serum for 6 h. [18F]FBNA partition coefficient (logP = 1.05) was found to be more lipophilic than [18F]EF-5 (logP = 0.75), [18F]FMISO (logP = 0.4) and [18F]FAZA (logP = - 0.4). In vitro studies showed that [18F]FBNA accumulates in gastric cancer cell lines AGS and MKN45 under hypoxic conditions. CONCLUSIONS: Hence, [18F]FBNA represents a novel and easy-to-prepare PET radioligand for imaging hypoxia.

Synthesis, Binding Affinity Analysis, and 18 F Radiosynthesis of Small-Molecular-Weight HIF-1α-Binding Compounds.

Eleven small-molecular-weight compounds and three cyclic peptides were synthesized and evaluated for binding to hypoxia-inducible factor-1α (HIF-1α). Microscale thermophoresis analysis identified peptide [19 F]SFB-link-c-(Ppg)LLFVY 3 and small-molecule inhibitor 5 as potent HIF-1α binding compounds with KD values of 0.46±0.2 µM and 7.8±3.4 µM, respectively. Both compounds represent novel HIF-1α-targeting compounds that are predicted to interact with the PAS-B region of HIF-1α, as confirmed with molecular docking studies. Lead structures 3 and 5 were further radiolabelled with fluorine-18 for positron emission tomography (PET) imaging agents targeting HIF-1α in vivo.

FOXM1 Inhibitors as Potential Diagnostic Agents: First Generation of a PET Probe Targeting FOXM1 To Detect Triple-Negative Breast Cancer in†vitro and in†vivo.

The FOXM1 protein controls the expression of essential genes related to cancer cell cycle progression, metastasis, and chemoresistance. We hypothesize that FOXM1 inhibitors could represent a novel approach to develop 18 F-based radiotracers for Positron Emission Tomography (PET). Therefore, in this report we describe the first attempt to use 18 F-labeled FOXM1 inhibitors to detect triple-negative breast cancer (TNBC). Briefly, we replaced the original amide group in the parent drug FDI-6 for a ketone group in the novel AF-FDI molecule, to carry out an aromatic nucleophilic (18 F)-fluorination. AF-FDI dissociated the FOXM1-DNA complex, decreased FOXM1 levels, and inhibited cell proliferation in a TNBC cell line (MDA-MB-231). [18 F]AF-FDI was internalized in MDA-MB-231 cells. Cell uptake inhibition experiments showed that AF-FDI and FDI-6 significantly decreased the maximum uptake of [18 F]AF-FDI, suggesting specificity towards FOXM1. [18 F]AF-FDI reached a tumor uptake of SUV=0.31 in MDA-MB-231 tumor-bearing mice and was metabolically stable 60 min post-injection. These results provide preliminary evidence supporting the potential role of FOXM1 to develop PET radiotracers.

Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles.

The presence of circulating tumor cells (CTCs) in a patient's bloodstream is a hallmark of metastatic cancer. The detection and analysis of CTCs is a promising diagnostic and prognostic strategy as they may carry useful genetic information from their derived primary tumor, and the enumeration of CTCs in the bloodstream has been known to scale with disease progression. However, the detection of CTCs is a highly challenging task owing to their sparse numbers in a background of billions of background blood cells. To effectively utilize CTCs, there is a need for an assay that can detect CTCs with high specificity and can locally enrich CTCs from a liquid biopsy. We demonstrate a versatile methodology that addresses these needs by utilizing a combination of nanoparticles. Enrichment is achieved using targeted magnetic nanoparticles and high specificity detection is achieved using a ratiometric detection approach utilizing multiplexed targeted and non-targeted surface-enhanced Raman Scattering Nanoparticles (SERS-NPs). We demonstrate this approach with model prostate and cervical circulating tumor cells and show the ex vivo utility of our methodology for the detection of PSMA or folate receptor over-expressing CTCs. Our approach allows for the mitigation of interference caused by the non-specific uptake of nanoparticles by other cells present in the bloodstream and our results from magnetically trapped CTCs reveal over a 2000% increase in targeted SERS-NP signal over non-specifically bound SERS-NPs.

A comparative PET imaging study of 44gSc- and 68Ga-labeled bombesin antagonist BBN2 derivatives in breast and prostate cancer models.

INTRODUCTION: Radiolabeled peptides play a central role in nuclear medicine as radiotheranostics for targeted imaging and therapy of cancer. We have recently proposed the use of metabolically stabilized GRPR antagonist BBN2 for radiolabeling with 18F and 68Ga and subsequent PET imaging of GRPRs in prostate cancer. The present work studied the impact of 44gSc- and 68Ga-labeled DOTA complexes attached to GRPR antagonist BBN2 on the in vitro GRPR binding affinity, and their biodistribution and tumor uptake profiles in MCF7 breast and PC3 prostate cancer models. METHODS: DOTA-Ava-BBN2 was radiolabeled with radiometals 68Ga and 44gSc. Gastrin-releasing peptide receptor (GRPR) affinities of peptides were assessed in PC3 prostate cancer cells. GRPR expression profiles were studied in human breast cancer tissue samples and MCF7 breast cancer cells. PET imaging of 68Ga- and 44gSc-labeled peptides was performed in MCF7 and PC3 xenografts as breast and prostate cancer models. RESULTS: Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava BBN2 were prepared in radiochemical yields of 70-80% (decay-corrected), respectively. High binding affinities were found for both peptides (IC50 = 15 nM (natGa) and 5 nM (natSc)). Gene expression microarray analysis revealed high GRPR mRNA expression levels in estrogen receptor (ER)-positive breast cancer, which was further confirmed with Western blot and immunohistochemistry. However, PET imaging showed only low tumor uptake of both radiotracers in MCF7 xenografts ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.27 ± 0.06); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.20 ± 0.03)). In contrast, high tumor uptake and retention were found for both radiopeptides in PC3 tumors ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.46 ± 0.07); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.51 ± 0.11)). CONCLUSIONS: Comparison of 68Ga- and 44gSc-labeled DOTA-Ava-BBN2 peptides revealed slight but noticeable differences of the radiometal with an impact on the in vitro GRPR receptor binding properties in PC3 cells. No differences were found in their in vivo biodistribution profiles in MCF7 and PC3 xenografts. Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava-BBN2 displayed comparable tumor uptake and retention profiles with rapid blood and renal clearance profiles in both tumor models. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: The favorable PET imaging performance of [44gSc]Sc-DOTA-Ava-BBN2 in prostate cancer should warrant the development of an [43Sc]Sc-DOTA-Ava-BBN2 analog for clinical translation which comes with a main γ-line of much lower energy and intensity compared to 44gSc.

Sulfo-click chemistry with 18F-labeled thio acids.

The first application of sulfo-click chemistry with 18F-labeled thio acids is described. The simple one-pot/three-component reaction proceeded rapidly within 30 min using mild reaction conditions to give various 18F-labeled small molecule N-acylsulfonamides in radiochemical conversions of 38-99%, and radiolabeled peptides in 20-25% isolated and decay-corrected radiochemical yields.

Impact of structural alterations on the radiopharmacological profile of 18F-labeled pyrimidines as cyclooxygenase-2 (COX-2) imaging agents.

INTRODUCTION: Non-invasive imaging of COX-2 in cancer represents a powerful tool for assessing COX-2-mediated effects on chemoprevention and radiosensitization using potent and selective COX-2 inhibitors as an emerging class of anticancer drugs. Careful assessment of the pharmacokinetic profile of radiolabeled COX-2 inhibitors is of crucial importance for the development of suitable radiotracers for COX-2 imaging in vivo. The delicate balance between the selection of typical COX-2 pharmacophores and the resulting physicochemical characteristics of the COX-2 inhibitor represents a formidable challenge for the search of radiolabeled COX-2 imaging agents. Several pyrimidine-based COX-2 inhibitors demonstrated favorable in vitro and in vivo COX-2 imaging properties in various COX-2 expressing cancer cell lines. Here, we describe a comparative radiopharmacological study of three 18F-labeled COX-2 inhibitors based on a pyrimidine scaffold. The objective of this study was to investigate how subtle structural alterations influence the pharmacokinetic profile of lead compound [18F]1a ([18F]Pyricoxib) to afford 18F-labeled pyrimidine-based COX-2 inhibitors with improved COX-2 imaging properties in vivo. METHODS: Radiosynthesis of radiotracers was accomplished through reaction with 4-[18F]fluorobenzyl amine on a methyl-sulfone labeling precursor ([18F]1a and [18F]2a) or late-stage radiofluorination using a iodyl-containing labeling precursor ([18F]3a). Radiopharmacological profile of 18F-labeled pyrimidine-based COX-2 inhibitors [18F]1a, [18F]2a and [18F]3a was studied in COX-2-expressing human HCA-7 colorectal cancer cell line, including cellular uptake studies in HCA-7 cells and dynamic PET imaging studies in HCA-7 xenografts. RESULTS: Cellular uptake of radiotracers [18F]2a and [18F]3a in HCA-7 cells was 450% and 300% radioactivity/mg protein, respectively, after 90 min incubation, compared to 600% radioactivity/mg protein for radiotracer [18F]1a. Dynamic PET imaging studies revealed a tumor SUV of 0.53 ([18F]2a) and 0.54 ([18F]3a) after 60 min p.i. with a tumor-to-muscle ratio of ~1. Tumor SUV for [18F]1a (60 min p.i.) was 0.76 and a tumor-to-muscle ratio of ~1.5. Pyricoxib analogues [18F]2a and [18F]3a showed distinct pharmacokinetic profiles in comparison to lead compound [18F]1a with a significantly improved lung clearance pattern. Replacing the 4-[18F]fluorobenzyl amine motif in radiotracer [18F]1a with a 4-[18F]fluorobenzyl alcohol motif in radiotracer [18F]3a resulted in re-routing of the metabolic pathway as demonstrated by a more rapid liver clearance and higher initial kidney uptake and more rapid kidney clearance compared to radiotracers [18F]1a and [18F]2a. Moreover, radiotracer [18F]3a displayed favorable rapid brain uptake and retention. CONCLUSION: The radiopharmacological profile of three 18F-labeled COX-2 inhibitors based on a pyrimidine scaffold were evaluated in COX-2 expressing human colorectal cancer cell line HCA-7 and HCA-7 xenografts in mice. Despite the overall structural similarity and comparable COX-2 inhibitory potency of all three radiotracers, subtle structural alterations led to significantly different in vitro and in vivo metabolic profiles. ADVANCES IN KNOWLEDGE: Among all tested pyrimidine-based 18F-labeled COX-2 inhibitors, lead compound [18F]1a remains the most suitable radiotracer for assessing COX-2 expression in vivo. Radiotracer [18F]3a showed significantly improved first pass pulmonary passage in comparison to radiotracer [18F]1a and might represents a promising lead compound for the development of radiotracers for PET imaging of COX-2 in neuroinflammation.

Targeting Prostate-Specific Membrane Antigen (PSMA) with F-18-Labeled Compounds: the Influence of Prosthetic Groups on Tumor Uptake and Clearance Profile.

PURPOSE: Prostate-specific membrane antigen (PSMA) is an important biomarker expressed in the majority of prostate cancers. The favorable positron emission tomography (PET) imaging profile of the PSMA imaging agent 2-(3-(1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl)-ureido)-pentane-dioic acid [18F]DCFPyL in preclinical prostate cancer models and in prostate cancer patients stimulated the development and validation of other fluorine-containing PSMA inhibitors to further enhance pharmacokinetics and simplify production methods. Here, we describe the synthesis and radiopharmacological evaluation of various F-18-labeled PSMA inhibitors which were prepared through different prosthetic group chemistry strategies. PROCEDURES: Prosthetic groups N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), 4-[18F]fluorobenzaldehyde, and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) were used for bioconjugation reactions to PSMA-binding lysine-urea-glutamate scaffold via acylation and oxime formation. All fluorine-containing PSMA inhibitors were tested for their PSMA inhibitory potency in an in vitro competitive binding assay in comparison to an established reference compound [125I]TAAG-PSMA. Tumor uptake and clearance profiles of three F-18-labeled PSMA inhibitors ([18F]4, [18F]7, and [18F]8) were studied with dynamic PET imaging using LNCaP tumor-bearing mice. RESULTS: F-18-labeled PSMA inhibitors were synthesized in 32-69 % radiochemical yields using (1) acylation reaction at the primary amino group of the lysine residue with [18F]SFB and (2) oxime formation with 4-[18F]fluorobenzaldehyde and [18F]FDG using the respective aminooxy-functionalized lysine residue. Compound 7 displayed an IC50 value of 6 nM reflecting very high affinity for PSMA. Compounds 4 and 8 showed IC50 values of 13 and 62 nM, respectively. The IC50 value of reference compound DCFPyL was 13 nM. Dynamic PET imaging revealed the following SUV60min for radiotracer uptake in PSMA(+) LNCaP tumors: 0.98 ([18F]DCFPyL), 2.11 ([18F]7), 0.40 ([18F]4), and 0.19 ([18F]8). CONCLUSION: The observed tumor uptake and clearance profiles demonstrate the importance of the selected prosthetic group on the pharmacokinetic profile of analyzed PSMA-targeting radiotracers. Radiotracer [18F]7 displayed the highest uptake and retention in LNCaP tumors, which exceeded uptake values of reference compound [18F]DCFPyL by more than 100 %. Despite the higher kidney and liver uptake and retention of compound [18F]7, the simple radiosynthesis and the exceptionally high tumor uptake (SUV60min 2.11) and retention make radiotracer [18F]7 an interesting alternative to radiotracer [18F]DCFPyL for PET imaging of PSMA in prostate cancer.

Targeting Phosphatidylserine with a 64Cu-Labeled Peptide for Molecular Imaging of Apoptosis.

Molecular imaging of programmed cell death (apoptosis) in vivo is an innovative strategy for early assessment of treatment response and treatment efficacy in cancer patients. Externalization of phosphatidylserine (PS) to the cell membrane surface of dying cells makes this phospholipid an attractive molecular target for the development of apoptosis imaging probes. In this study, we have radiolabeled PS-binding 14-mer peptide FNFRLKAGAKIRFG (PSBP-6) with positron-emitter copper-64 (64Cu) for PET imaging of apoptosis. Peptide PSBP-6 was conjugated with radiometal chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through an aminovaleric acid (Ava) linker for subsequent radiolabeling with 64Cu to prepare radiotracer 64Cu-NOTA-Ava-PSBP-6. PS-binding potencies of PSBP-6, NOTA-Ava-PSBP-6, and natCu-NOTA-Ava-PSBP-6 were determined in a competitive radiometric PS-binding assay. Radiotracer 64Cu-NOTA-Ava-PSBP-6 was studied in camptothecin-induced apoptotic EL4 mouse lymphoma cells and in a murine EL4 tumor model of apoptosis using dynamic PET imaging. Peptide PSBP-6 was also conjugated via an Ava linker with fluorescein isothiocyanate (FITC). FITC-Ava-PSBP-6 was evaluated in flow cytometry and fluorescence confocal microscopy experiments. Radiopeptide 64Cu-NOTA-Ava-PSBP-6 was synthesized in high radiochemical yields of >95%. The IC50 values for PS-binding potency of PSBP-6, NOTA-Ava-PSBP-6, and natCu-NOTA-PSBP-6 were 600 µM, 30 µM, and 23 µM, respectively. A competitive radiometric cell binding assay confirmed binding of 64Cu-NOTA-Ava-PSBP-6 to camptothecin-induced apoptotic EL4 cells in a Ca2+-independent manner. PET imaging studies demonstrated significantly higher uptake of 64Cu-NOTA-Ava-PSBP-6 in apoptotic EL4 tumors (SUV5min 0.95 ± 0.04) compared to control tumors (SUV5min 0.74 ± 0.03). Flow cytometry studies showed significantly higher binding of FITC-Ava-PSBP-6 to EL4 cells treated with camptothecin compared to untreated cells. Fluorescence microscopy studies revealed that FITC-Ava-PSBP-6 was binding to cell membranes of early apoptotic cells, but was internalized in late apoptotic and necrotic cells. The present study showed that radiotracer 64Cu-NOTA-Ava-PSBP-6 holds promise as a first peptide-based PET imaging agent for molecular imaging of apoptosis. However, additional "fine-tuning" of 64Cu-NOTA-Ava-PSBP-6 is required to enhance PS-binding potency and in vivo stability to improve tumor uptake and retention.

PET imaging of cyclooxygenase-2 (COX-2) in a pre-clinical colorectal cancer model.

BACKGROUND: Cyclooxygenase-2 (COX-2) is the inducible isoform of the cyclooxygenase enzyme family. COX-2 is involved in tumor development and progression, and frequent overexpression of COX-2 in a variety of human cancers has made COX-2 an important drug target for cancer treatment. Non-invasive imaging of COX-2 expression in cancer would be useful for assessing COX-2-mediated effects on chemoprevention and radiosensitization using COX-2 inhibitors as an emerging class of anti-cancer drugs, especially for colorectal cancer. Herein, we describe the radiopharmacological analysis of [(18)F]Pyricoxib, a novel radiolabeled COX-2 inhibitor, for specific PET imaging of COX-2 in colorectal cancer. METHODS: Uptake of [(18)F]Pyricoxib was assessed in human colorectal cancer cell lines HCA-7 (COX-2 positive) and HCT-116 (COX-2 negative). Standard COX-2 inhibitors were used to test for specificity of [(18)F]Pyricoxib for COX-2 binding in vitro and in vivo. PET imaging, biodistribution, and radiometabolite analyses were included into radiopharmacological evaluation of [(18)F]Pyricoxib. RESULTS: Radiotracer uptake in COX-2 positive HCA-7 cells was significantly higher than in COX-2 negative HCT-116 cells (P < 0.05). COX-2 inhibitors, celecoxib, rofecoxib, and SC58125, blocked uptake of [(18)F]Pyricoxib in HCA-7 cells in a concentration-dependent manner. The radiotracer was slowly metabolized in mice, with approximately 60 % of intact compound after 2 h post-injection. Selective COX-2-mediated tumor uptake of [(18)F]Pyricoxib in HCA-7 xenografts was confirmed in vivo. Celecoxib (100 mg/kg) selectively blocked tumor uptake by 16 % (PET image analysis; P < 0.05) and by 51 % (biodistribution studies; P < 0.01). CONCLUSIONS: The novel PET radiotracer [(18)F]Pyricoxib displays a promising radiopharmacological profile to study COX-2 expression in cancer in vivo.

Metabolically Stabilized (68)Ga-NOTA-Bombesin for PET Imaging of Prostate Cancer and Influence of Protease Inhibitor Phosphoramidon.

Peptide receptor-based targeted molecular imaging and therapy of cancer is on the current forefront of nuclear medicine preclinical research and clinical practice. The frequent overexpression of gastrin-releasing peptide (GRP) receptors in prostate cancer stimulated the development of radiolabeled bombesin derivatives as high affinity peptide ligands for selective targeting of the GRP receptor. In this study, we have evaluated a novel (68)Ga-labeled bombesin derivative for PET imaging of prostate cancer in vivo. In addition, we were interested in testing the recently proposed "serve-and-protect" strategy to improve metabolic stability of radiolabeled peptides in vivo and to enhance tumor uptake. GRP receptor targeting peptides NOTA-BBN2 and (nat)Ga-NOTA-BBN2 demonstrated a characteristic antagonistic profile and high binding affinity toward the GRP receptor in PC3 cells (IC50 4.6-8.2 nM). Radiolabeled peptide (68)Ga-NOTA-BBN2 was obtained from NOTA-BBN2 in radiochemical yields greater than 62% (decay-corrected). Total synthesis time was 35 min, including purification using solid-phase extraction. (68)Ga-NOTA-BBN2 exhibited favorable resistance against metabolic degradation by peptidases in vivo within the investigated time frame of 60 min. Interestingly, metabolic stability was not further enhanced in the presence of protease inhibitor phosphoramidon. Dynamic PET studies showed high tumor uptake in both PC3- and LNCaP-bearing BALB/c nude mice (SUV5min > 0.6; SUV60min > 0.5). Radiotracer (68)Ga-NOTA-BBN2 represents a novel radiometal-based bombesin derivative suitable for GRP receptor targeting in PC3 and LNCaP mouse xenografts. Further increase of metabolic stability in vivo and enhanced tumor uptake were not observed upon administration of protease inhibitor phosphoramidon. This led to the conclusion that the recently proposed "serve-and-protect" strategy may not be valid for peptides exhibiting favorable intrinsic metabolic stability in vivo.

Radiopharmacological evaluation of (18)F-labeled phosphatidylserine-binding peptides for molecular imaging of apoptosis.

INTRODUCTION: Radiolabeled phosphatidylserine (PS)-binding peptides represent an innovative strategy for molecular imaging of apoptosis with positron emission tomography (PET). The goal of this study was the radiopharmacological evaluation of radiolabeled peptides for their binding to PS on apoptotic cancer cells, involving metabolic stability, cellular uptake, biodistribution, and dynamic PET imaging experiments. METHODS: Binding of peptides LIKKPF, PGDLSR, FBz-LIKKPF, FBz-PGDLSR, FBAM-CLIKKPF and FBAM-CPGDLSR to PS was analyzed in a newly developed radiometric binding assay using (64)Cu-labeled wild-type annexin-V as radiotracer. Radiolabeling of most potent peptides with fluorine-18 was carried out with thiol-selective prosthetic group [(18)F]FBAM to give [(18)F]FBAM-CLIKKPF and [(18)F]FBAM-CPGDLSR. [(18)F]FBAM-labeled peptides were studied in camptothecin-induced apoptotic human T lymphocyte Jurkat cells, and in a murine EL4 tumor model of apoptosis using dynamic PET imaging and biodistribution. RESULTS: Peptides LIKKPF and PGDLSR inhibited binding of (64)Cu-labeled annexin-V to immobilized PS in the millimolar range (IC50 10-15 mM) compared to annexin-V (45 nM). Introduction of FBAM prosthetic group slightly increased inhibitory potencies (FBAM-CLIKKPF: IC50 = 1 mM; FBAM-CPGDLSR: IC50 = 6 mM). Radiolabeling succeeded in good radiochemical yields of 50-54% using a chemoselective alkylation reaction of peptides CLIKKPF and CPGDLSR with [(18)F]FBAM. In vivo metabolic stability studies in mice revealed 40-60% of intact peptides at 5 min p.i. decreasing to 25% for [(18)F]FBAM-CLIKKPF and less than 5% for [(18)F]FBAM-CPGDLSR at 15 min p.i.. Cell binding of [(18)F]FBAM-CLIKKPF in drug-treated Jurkat cells was significantly higher compared to untreated cells, but this was not observed for [(18)F]FBAM-CPGDLSR. Dynamic PET imaging experiments showed that baseline uptake of [(18)F]FBAM-CLIKKPF in EL4 tumors was higher (SUV(5min) 0.46, SUV(60min) 0.13) compared to [(18)F]FBAM-CPGDLSR (SUV(5min) 0.16, SUV(60min) 0.10). Drug-treated EL4 tumors did not show an increased uptake for both [(18)F]FBAM-labeled peptides. CONCLUSION: Although both (18)F-labeled peptides [(18)F]FBAM-CLIKKPF and [(18)F]FBAM-CPGDLSR showed higher binding to apoptotic Jurkat cells in vitro, their in vivo uptake profiles were not different in apoptotic EL4 tumors. This may explained by the relatively low potency of both compounds to compete with binding of (64)Cu-labeled annexin-V to PS. Overall the novel competitive radiometric PS-binding assay with (64)Cu-labeled annexin-V represents a versatile and very robust screening platform to analyze potential PS-binding compounds in vitro. Further studies will be necessary to evaluate alternative peptide structures toward their use as PET radiotracers imaging apoptosis in vivo. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Development of peptide-based radiotracers for imaging apoptosis in vivo remains a significant challenge.

Sonogashira cross-coupling reaction with 4-[18F]fluoroiodobenzene for rapid 18F-labelling of peptides.

The study describes the Sonogashira cross-coupling reaction with 4-[(18)F]fluoroiodobenzene ([(18)F]FIB) as novel and efficient method for rapid labelling of peptides with the short-lived positron emitter fluorine-18.

Rerouting the metabolic pathway of (18)F-labeled peptides: the influence of prosthetic groups.

Current translational cancer research is directed to the development of high affinity peptide ligands for targeting neuropeptide receptors overexpressed in different types of cancer. Besides their desired high binding affinity to the receptor, the suitability of radiolabeled peptides as targeting vectors for molecular imaging and therapy depends on additional aspects such as high tumor-to-background ratio, favorable clearance pattern from nontarget tissue, and sufficient metabolic stability in vivo. This study reports how a switch from the prosthetic group, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB), to 2-deoxy-2-[(18)F]fluoro-d-glucose ([(18)F]FDG) effects the metabolic pathway of an (18)F-labeled bombesin derivative, QWAV-Sar-H-FA01010-Tle-NH2. (18)F-Labeled bombesin derivatives represent potent peptide ligands for selective targeting of gastrin-releasing peptide (GRP) receptor-expressing prostate cancer. Radiosynthesis of (18)F-labeled bombesin analogues [(18)F]FBz-Ava-BBN2 and [(18)F]FDG-AOAc-BBN2 was achieved in good radiochemical yields of ~50% at a specific activity exceeding 40 GBq/µmol. Both nonradioactive compounds FBz-Ava-BBN2 and FDG-AOAc-BBN2 inhibited binding of [(125)I]Tyr(4)-bombesin(1-14) in PC3 cells with IC50 values of 9 and 16 nM, respectively, indicating high inhibitory potency. Influence of each prosthetic group was further investigated in PC3 mouse xenografts using dynamic small animal PET imaging. In comparison to [(18)F]FBz-Ava-BBN2, total tumor uptake levels were doubled after injection of [(18)F]FDG-AOAc-BBN2 while renal elimination was increased. Blood clearance and in vivo metabolic stability were similar for both compounds. The switch from [(18)F]SFB to [(18)F]FDG as the prosthetic group led to a significant reduction in lipophilicity which resulted in more favorable renal clearance and increased tumor uptake. The presented single step radiolabeling-glycosylation approach represents an innovative strategy for site-directed peptide labeling with the short-lived positron emitter (18)F while providing a favorable pharmacokinetic profile of (18)F-labeled peptides.